The impact of MYD88 and PIM1 in mature large B-cell non-Hodgkin lymphomas: Defining element of their evolution and prognosis

Sequence studies of the entire exome and transcriptome of lymphoma tissues have identified MYD88 and PIM1 as involved in the development and oncogenic signaling. We aimed to determine the frequency of MYD88 and PIM1 mutations, as well as their expressions in conjunction with the clinicopathological parameters identified in mature large B-cell non-Hodgkin lymphomas. The ten-year retrospective study included 50 cases of mature large B-cell lymphoma, diagnosed at the Pathology Department of the Emergency County Hospital of Constanţa and Săcele County Hospital of Brasov. They were statistically analyzed by demographic, clinicopathological, and morphogenetic characteristics. We used a real-time polymerase chain reaction technique to identify PIM1 and MYD88 mutations as well as an immunohistochemical technique to evaluate the expressions of the 2 genes. Patients with lymphoma in the small bowel, spleen, brain, and testis had a low-performance status Eastern Cooperative Oncology Group (P = .001). The Eastern Cooperative Oncology Group performance status represented an independent risk factor predicting mortality (HR = 9.372, P < .001). An increased lactate dehydrogenase value was associated with a low survival (P = .002). The international prognostic index score represents a negative risk factor in terms of patient survival (HR = 4.654, P < .001). In cases of diffuse large B-cell lymphoma (DLBCL), immunopositivity of MYD88 is associated with non-germinal center B-cell origin (P < .001). The multivariate analysis observed the association between high lactate dehydrogenase value and the immunohistochemical expression of PIM1 or with the mutant status of the PIM1 gene representing negative prognostic factors (HR = 2.066, P = .042, respectively HR = 3.100, P = .004). In conclusion, our preliminary data suggest that the oncogenic mutations of PIM1 and MYD88 in our DLBCL cohort may improve the diagnosis and prognosis of DLBCL patients in an advanced stage.


Introduction
Non-Hodgkin lymphoma (NHL) is a heterogeneous group of tumors that arise from developing lymphocytes and is far more common than Hodgkin lymphoma. [1,2]The most common NHL subtype in South-Eastern Europe was diffuse large B-cell lymphoma (DLBCL) (39%), followed by follicular lymphoma (15,8%) and the relative frequency of DLBCL is significantly higher in South-Eastern Europe compared to Western Europe (WEU) (29,3%) and North America (NA) (28.3%). [3]he family/class of large B-cell lymphomas comprises a spectrum of tumors with varying morphologies, genetic features, and clinical behavior.Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS), represents the largest entity, and is defined by morphology and a mature B-cell phenotype. [4]iffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma, and despite advances in survival with the introduction of rituximab, about one-third of patients with advanced stage are still unresponsive to current therapy or eventually relapse. [5,6]ost DLBCL, NOS broadly recapitulate the differentiation and maturation mechanisms active in normal B cell development and hence, 2 main subtypes previously defined in the World Health Organization (WHO) 4th edition (revised) continue to be recognized-the germinal center B-cell subtype (GCB) and the activated B-cell subtype (ABC).More recent data from next-generation sequencing studies have illustrated that-despite the use of different sequencing approaches and various clustering algorithms-the genetic landscape of DLBCL, NOS can be used for sub-classification with broad concordance suggesting that the underlying disease biology may indeed be captured by comprehensive genetic landscapes. [4]ymphoma types/entities are defined by morphological, immunophenotypical, and clinical criteria, but none of these are absolutely specific and defining. [4]he morphological and/or clinical settings defining the entity/type, respectively, overrule the genetic characteristics in such cases.Similarly, an immune deficiency/dysregulation setting overrules other defining parameters (e.g., central nervous system [CNS] localization of immune deficiency/dysregulationassociated DLBCL, Epstein-Barr virus-positive, human immunodeficiency virus [HIV]-positive setting). [4]hey are enriched for B-cell antigen receptor pathway mutations such as in MYD88, CD79B, provirus integration site for Moloney murine leukemia virus 1 (PIM1), and PRDM1 encoding BLIMP1. [7]he myeloid differentiation primary response 88 (MYD88) protein is an important adaptor molecule in Toll-like receptors signaling that prompts the activation of NF-kB and the production of both inflammatory cytokines and type I interferons. [8]][11][12] Ngo et al found that 29% of ABC DLBCL patients had a leucine (CTG) to proline (CCG) ex-change at position 265 (L265P) of the myeloid differentiation main response gene 88, which may be another factor contributing to NF-B overactivity.Therapeutically, inhibiting NF-B signaling may be used to treat DLBCL, which is not compliant. [5,9]he identification as cooperating targets of Proviral Integrations of Moloney virus in murine lymphomas suggested early on that PIM serine/threonine kinases play an important role in cancer biology.Elevated levels of PIM1 were mostly found in hematological malignancies.PIM1 seems to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression.Knockdown experiments by ribonucleic acid interference or dominant-negative acting mutants suggested that PIM kinases are important for the maintenance of a transformed phenotype and therefore potential therapeutic targets. [13]IM1 expression is correlated with poor prognosis in DLBCL, NOS and the most common PIM1 mutations identified in patients with poor response to targeted therapy are G28D, L2V, and S97N.[14,15] Therefore, PIM1 appears as an attractive target in the therapy of hematopoietic neoplasms and as a biomarker of early progression.
However, the MYD88 L265P and PIM1 p.G28A, p.L184V, and p.V197F mutations have not yet been examined in conjunction with MYD88 and PIM1 protein expression in large B-cell NHL cases.
In this study, we aimed to determine the MYD88 L265P and PIM1 p.G28A, p.L184V, p.V197F mutation frequency, the level of MYD88 and PIM1 immunohistochemical expression, and their associations with each other and with clinicopathological parameters among patients with large B-cell non-Hodgkin lymphomas in Romania.
Therefore, it may be essential to perform a determination of these mutations and, if necessary, categorize the tumors in light of the existence of the mutation in lymphomas.

Patient cohort and tissue tumor specimens
We performed a retrospective study over 10 years (2012-2021) that included cases of mature large B-cell NHL.The cases enrolled in this study were diagnosed in the hematology department of the County Emergency Clinical Hospital of Constanţa and Sacele County Hospital of Brasov.The hospital's archives and electronic databases were used to extract the data.The patient's clinical information and evolutionary information came from the hospitalization electronic medical records.The data were evaluated by the attending physician and the hematologist.
Following the surgical procedures, the surgical specimens were sent to the pathology department of the same hospital for examination.First, a gross description was made, along with information about the lesions' largest diameter, localization, color, and consistency of specimens.The specimens were prepared and processed according to international protocols within the Clinical Service of Anatomic Pathology, County Emergency Hospital, Constanta, and Service of Anatomic Pathology, Sacele Municipal Hospital, Brasov, and by embedding them in paraffin and applying hematoxylin-eosin on the slides.Consecutive sections of formalin-fixed and paraffin-embedded (FFPE) tissue were obtained for deoxyribonucleic acid (DNA) isolation and immunohistochemistry (IHC) analysis.
The final diagnosis was made according to the WHO criteria appropriate to the year in which the patients were diagnosed.Two different pathologists reevaluated the relevant cases while taking into account the most recent WHO classification guidelines (5th edition) and following the guidelines of the International Consensus Classification of Mature Lymphoid Neoplasms. [4,16]ll patients were classified according to the Eastern Cooperative Oncology Group (ECOG) Performance Status, Ann Arbor staging system, and International Prognostic Index (IPI) score, following the criteria described in previous studies by Lister et al [17,18] The classification of tumors as GCB-like and as the non-GCB subtype in DLBCL was performed according to the algorithm of Hans et al (2004). [19]he diagnosis of high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS) was made by histological features and immunohistochemical examination.
The scanning of the slides was performed at the Center for Research and Development of the Morphological and Genetic Studies of Malignant Pathology (CEDMOG).The WSIs were obtained using the Huron LE120TM 4000XT scanner (Huron Technolo-gies International Inc., Canada).www.md-journal.comTissue samples were collected from all patients before treatment.
Our study was approved by the Ethics Committee of both hospitals and all the patients signed the informed consent at the time of hospitalization and, conducted in compliance with the Declaration of Helsinki.
Tissue blocks provided by the Clinical Service of Pathology of the Clinical Emergency County Hospital "Sf.Apostol Andrei" and Municipal Hospital of Sacele were cut into 4 µm sections and applied on a slide.
Positive controls, generated by replacing the primary antibody with phosphate-buffered saline, made by using samples of kidney tissue for MYD88 and tonsils tissue for PIM1, that had previously been evaluated, were utilized in each staining run.The number of MYD88-and, PIM1-immunoreactive cells in at least 10 areas (hot spot method) was determined using a 40× objective lens and a light microscope.
The staining protocol followed the producer's recommendations: for deparaffinized, we used xylene and decreasing grades of alcohol.The Novus Biologicals protocol for PIM1 antibody (ST0513; [NBP2-67528]) and MYD88 antibody (RM306; [NBP2-61565]) immunostaining included HIER in citrate pH 6.0 buffer and incubation overnight at 4 °C with a 1:200 dilution for PIM1 and1:150 for MYD88.The detection stage required a polymer detection kit that included peroxidase, 2.5% normal horse serum, HRP, and DAB reagent-Vector Laboratories, Burlingame, CA.In the final stage, we counterstained with hematoxylin and mounted the glass cover slides.
The evaluation of PIM1 and MYD88 was carried out qualitatively as well as quantitatively.The quality of the expression was assessed as positive or negative, and the amount of PIM1 and MYD88 immunoreactive cells in at least 10 areas was determined using a 40× objective lens and a light microscope.
According to the data presented in a prior study by Choi et al, [5] the following immunohistochemistry scoring system was used to evaluate MYD88 immunohistochemical staining.Scores for staining intensity ranged from 0 (negative), 1 (weak), 2 (moderate), or 3 (intense) and the extent of staining was scored as 0 (0% of tumor area stained), 1 (<10%), 2 (10-50%), or 3 (˃50%).Each example received a total score ranging from 0 to 6 once these 2 scores were added together.The tissue microarray core with the highest score was chosen for statistical analysis in each case.A low and high MYD88 expression, respectively, was determined by the final scores of 2 to 4 and 5 to 6.

DNA isolation
Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) according to the manufacturer's protocol.In brief, tissue areas up to 250 mm 2 and 8 sections with a maximum thickness of 10 mm were used for DNA isolation.Hematoxylin and eosin-stained sections were used as a reference and the largest tumor area (with at least 50% tumor cells) was scraped off with a scalpel under a dissecting microscope.The concentration and purity of DNA samples were measured by Nanodrop One spectrophotometer, where an absorbance ratio A260/A280 = 1.8-2.0,and A260/A230 > 2 was considered acceptable.

Genotyping of the MYD88 (rs387907272, T/C) and PIM1 (rs377274719, G/A; rs200495767 C/G; rs137884665 G/T) polymorphisms
SNPs polymorphisms of the MYD88 and PIM1 were identified using a real-time PCR method based on the readymade TaqMan® Genotyping Master Mix and 20× SNP Genotyping Assay (specific for each polymorphism) containing targetspecific oligo-nucleotides labeled with a reporter dye at the 5´ end of each probe for distinguishing between the 2 alleles: VIC dye is linked to the 5´ end of the Allele 1 probe and 6FAM dye is linked to the 5´ end of the Allele 2 probe.(Table 1).The DNA concentration was set between 1 to 10 ng per 10 μL of realtime polymerase chain reaction.Shortly, each 10 μL real-time polymerase chain reaction consists of 5 μL TaqMan Genotyping Master Mix (2×), 0.5 μL of TaqMan genotyping assay mix (20×), and a 4.5 μL DNA.Samples were incubated in a 7500 Fast Real-Time System with the following parameters: 95 °C for 10 minutes, 95 °C for 15 seconds, and 60 °C for 1 minute.The last 2 steps of denaturing and annealing/extending were repeated 40 times.Allelic discrimination was made with the help of 7500 Fast Real-Time PCR software, version 2.3.

Statistical analysis
SPSS Statistics Version 26 (IBM Corporation, NY) was used to analyze statistical data.Both central tendency and variability indicators were applied.For categorical data, Fisher exact test, and for continuous variables, the Mann-Whitney U test and Kruskal-Wallis H test, were used in an analysis of univariate data.To establish the association of data, we used the Pearson correlation coefficient.Survival estimates were performed until March 1, 2023, and they were calculated using the Kaplan-Meier method.Applying the log-rank test, the survival differences between groups were examined.The use of Cox regression analysis allowed for the appreciation of hazard ratios (HRs).Results were considered statistically significant at a P-value of <.05.

Table 1
SNPs polymorphisms of the MYD88 and PIM1.

Patient number
Rs Genes Sequence VIC/FAM Mature large B-cell lymphomas affected both sexes equally (M:F-1:1).The average age of the patients was 61.04 years (27-101 years), with the majority of patients being over 60 years old (60%).More than half of the cases were found in the lymph nodes (54%).The other main localizations of large B-cells NHL, according to the analysis of the studied batch, are the gastrointestinal tract (34%) and CNS (8%).Also, the most frequent localization in the GI tract was in the stomach (10%) and the small intestine (10%).
Patients with lymphoma presented a low ECOG performance score (0-1) at admission in most of the cases (70%).According to the patient's hospitalization medical records, the most frequently reported comorbidities were secondary anemia (74%), hypertension (20%), and diabetes mellitus (14%).The advanced age of the patients, especially those over 60 years, was associated with the presence of high blood pressure (P = .038,respectively P = .031).Also, an association with the presence of atrial fibrillation was observed in elderly patients (P = .016).The presence of dyslipidemia was observed especially in the case of DLBCL 5 of 40 (12,5%), showing significant statistical associations with the female sex (P = .018).Hypertension was found in close association with hyperuricemia (P = .020),atrial fibrillation (P < .001),hematuria (P = .002),renal failure (P = .003),upper digestive hemorrhage (P = .003)and cardiac insufficiency (P < .001).
Some of the patients presented antecedents of acute infections in 16% of cases (with Klebsiella pneumoniae, Staphylococcus epidermidis, Streptococcus, Bacillus spp., Pseudomonas aeruginosa; Helicobacter pylori, and Candida albicans) or chronic infections like hepatitis virus C (8%), tuberculosis (8%), hepatitis virus B (4 %) or HIV (4%).The patients with HIV infection were younger than the others without HIV (P = .003).A predominance of male patients has been observed in the distribution of infectious disease antecedents (P = .007)and the hepatitis virus C was observed mostly in female patients (P = .038).
From a biochemical point of view, the most important change was the increase in lactate dehydrogenase (LDH) values (56% of cases).It had an average value of 471.04 IU/L.In the case of anemic syndrome, the average value of hemoglobin was 10.93 g/ dl (4.90-15.70g/dL).Regarding the association between the laboratory tests, we observed an increased LDH at admission that was statistically significantly associated with the presence of anemia (P = .033),and also with low hemoglobin values (P = .011).Also, we observed a statistically significant association between the LDH values and hemoglobin-the higher the LDH value, the lower the hemoglobin (P = .001).However, in the case of increased LDH values, the ECOG performance status was low (0-1), (P = .033).In the case of hemoglobin, its decrease was also associated with the presence of upper digestive hemorrhage (P = .021),as well as with other concomitant neoplasms (P = .046).An increased LDH value was associated with low survival of the patients (P = .002),869.34 days (124.19 weeks) versus 2447.10 days (349.58 weeks), (Fig. 1).An elevated LDH value at admission represented an independent negative risk factor in terms of patient survival (HR = 3.100, P = .004)(Fig. 2 and Table 2).
In most cases, an average percentage of lymphocytes of 2.74% (0.34-22.95%) was observed in the peripheral blood, with a value of 21.53 × 10 3 /L of lymphocytes.
Depending on the localizations of the lymphoma, the testis or spleen was associated with the lowest survival, 7 days (one week), respectively 49.14 days (7.02 weeks), (Fig. 3).Also, lower survival was observed in the extranodal localization of lymphomas compared with nodal localization of lymphomas (P = .130)(Fig. 4).
Ann Arbor staging highlighted a slight majority of cases (52%) in the low-grade category (I-II).The same aspect was found and could be transposed in the international prognostic index score, with 64% of the cases being at low risk.A lowgrade Ann Arbor staging was associated with nodal localizations (P < .001),and a similar aspect was also noted in the cases with a low IPI score (P = .001).A high IPI score was associated with extranodal localizations other than those in the skin or Figure 1.Kaplan-Meier survival graphic that shows a lower survival associated with an increased LDH value at the presentation of the patients (P = .002).www.md-journal.comlarge bowel (P < .001).Also, a high IPI score was found in cases of elevated LDH values (P = .001).
Unfortunately, not all patients benefited from the treatment, but only 62% of them, and the preferred scheme was rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) (44%).This treatment scheme was especially associated with a low age of the patients (P = .034),but also with the nodal localization of the lymphoma (P = .004).Both R-CHOP and cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) protocol schemes were associated with a good performance status ECOG (P < .001),with a low Ann Arbor stage (P = .005),and with a favorable IPI risk score (P < .001).
At the end of this study, only 36% of patients were alive.The average survival was 913.42 days (130.48 weeks).Patients with a good performance status (0-1) had much higher average survival, 2176.49days (310.92weeks) vs. 54.36days (7.76 weeks), compared to those with an altered status (P < .001).The ECOG performance status represented an independent risk factor predicting mortality (HR = 9.372, P < .001)(Fig. 5 and Table 2).
The most effective treatment for survival was the use of the CHOP scheme, in its case an average survival of 2323.32 days (331.90 weeks) was observed, while in the R-CHOP scheme, an average of 2278.90 days (325.55 weeks) was observed, and those who did not perform chemotherapy treatment had an average of 117.16 days (16.73 weeks), (P < .001).The lack of chemotherapeutic treatment represents a risk factor regarding patient mortality (HR = 6.750,P < .001)(Table 2).The IPI score was associated with patient survival, so those with high risk had a lower average, 54.36 days (7.76 weeks) versus 2176.49days (310.92weeks), compared to those in the low-risk category (P < .001).The high risk represented by the IPI score represents a negative risk factor in terms of patient survival (HR = 4.654, P < .001)(Fig. 6 and Table 2).
The multivariate analysis of the data shows that the associations between a low-performance status (2-4) with the age of over 60 and an increased LDH with the presence of anemia are risk factors regarding survival (HR = 7.715, P < .001,respectively HR = 3.582, P = .020)(see Table 2).
The distribution of demographic aspects, tumor characteristics, and personal history are stratified according to diagnosis in Table 3.

MYD88 L265P and MYD88 expression
Genetic analysis revealed a mutant status of MYD88 in 8% of cases and of PIM1 in 2% of cases (Table 4).MYD88 L265P mutation was observed in 4 of 40 (8%) of DLBCL cases.
Patients with the L265P mutation were 1 man and 3 women, with a median age of 68.5 years, ranging from 54 to 82 years.Three patients were older than 60 years.None of the patients with the L265P mutation showed a significant association with clinical parameters of DLBCL, including the patient's age, sex, tumor location, ECOG performance status, LDH level, IPI score, and Ann Arbor stage.All but 2 cases were in the non-GCB subgroup.
MYD88 was expressed in lymphoma cells in 8 (16%) of 50 cases.MYD88 was expressed in the cytoplasm of the lymphoid cells (Fig. 7A and C).
In MYD88 immunopositivity, the quantification of the reaction revealed a low score (2-4) in 10% of cases and a high score (5-6) in 6% of cases.A high score was associated with HGBL lymphomas, while a low score was correlated with THRLBCL and primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) (P = .005).In cases of DLBCL, immunopositivity quantified as low and high score is associated with non-GCB origin (P < .001),but not with other clinicopathologic parameters.Three patients died of the disease, and one of them survived and showed a low MYD88 expression score.Increased survival was observed for wild-type status, 1574.96days (224.99 weeks), as opposed to mutant status, 695.31 days (99.33 weeks), with no statistically significant significance (P = .505).
PIM1 was expressed in lymphoma cells in 27 (54%) of 50 cases (Table 4).PIM1 was expressed in the nuclei of the lymphoid cells (Fig. 7B and D).
None of the patients with the PIM1 p.G28A mutation showed a significant association with clinical parameters, including the patient's age, sex, tumor location, ECOG performance status, LDH level, IPI score, and Ann Arbor stage.
The multivariate analysis observed that the association between a high LDH at admission and the immunohistochemical expression of PIM1 or with the mutant status of the PIM1 gene, represent negative prognostic factors (HR = 2.066, P = .042,respectively HR = 3.100, P = .004)(see Table 2).
No statistically significant correlation was observed between PIM1 expression and PIM1 p.G28A mutation (Spearman ρ = −0.132,P = .361).In the case of mutant status, no deaths were observed, these being noted only in the wild-type status.

Discussion
With an estimated 150,000 new cases each year globally, large B-cell lymphomas account for over 30% of all non-Hodgkin  lymphoma cases. [4,20]The updated WHO classification has revised the categorization of large B-cell lymphomas, which are a heterogeneous group of clinicopathological entities that diffuse large B-cell lymphoma, not otherwise specified, is the most common. [20]In the study we performed, DLBCL with NOS was the most prevalent in the group of patients examined (80%), and the findings were in agreement with the data provided by WHO 2022.
The majority of patients with DLBCL, NOS present with nodal disease, and around 30% to 40% of cases present with disease confined to extranodal sites at diagnosis. [4]Virtually any site can be involved but common extranodal sites include the gastrointestinal tract (stomach and ileocecal region). [4]onsistent with those findings, we identified that half of the cases of DLBCL, NOS were found in the lymph nodes 20 of 40 (50%).[23][24] In our research, the most frequent extranodal localizations of the lymphomas were gastrointestinal tract (34%), the majority being represented by the stomach (10%), small bowel (10%), and large bowel (6%).
The average age of DLBCL at diagnosis, according to Sehn et al, is in the middle of the 60s, and 30% of patients are over 75 years. [20]In our study, the average age of the patients with  DLBCL, NOS at diagnosis was similar to the previous estimate (60.78 years).
Limited data are available specifically for HGBL, NOS but at least it is established that the incidence generally increases with age and, consequently, elderly patients are affected most often. [4]In our study, the average age of the patients diagnosed with HGBL, NOS was under those values (55.25 years), underlying the predominance of the disease in younger patients.
THRLBCL accounts for <10% of DLBCL and it preferentially affects middle-aged or older adults (age range 18-90  years), with rare cases affecting children. [4]In our study, the median age of the patients diagnosed with THRLBCL was similar to those values (58 years).PCLBCL-LT typically occurs in elderly patients with a median age of around 75 years, [4] but in our study, the median age of the patients with PCLBCL-LT was more than those values (84 years).
A definitive diagnosis of DLBCL is necessary according to the WHO classification of Tumors of Hematopoietic and Lymphoid Tissues to obtain data regarding the COO, which may be determined using immunohistochemistry (IHC) stainings or gene expression profiling (GEP). [4]Regarding the molecular subtypes, the frequency of the GCB subtype is around 60%, whereas that of the ABC subtype is between 25% and 30%. [20]Our research identified the cases where the COO could be identified (80%), with a higher predominance of the GCB subtype (64%) and a lower predominance of the non-GCB subtype (16%).
DLBCL, NOS occurs in men slightly more frequently than in women. [4]Similarly, in our case, the male sex diagnosed with DLBCL, NOS is the most affected, observed in 21 of 40 (52.5%) of cases.
In high-grade B-cell lymphoma, NOS cases no sex predilection has been reported.In our research, females are the most affected 3 of 4 (75%).
PCLBCL-LT is more common in women, with a female-tomale ratio between 2:1 and 4:1. [4]In contrast, in our study, PCLBCL-LT affects both sexes equally (M:F = 1:1).R-CHOP therapy is currently the gold standard of care for DLBCL patients across the world. [25,26]However, 20% to 50% of DLBCL patients relapse or are resistant to first-line therapy, making them eligible for second-line and later treatments. [27,28]nfortunately, there have traditionally been few therapies available once a patient has reached the third line, and overall survival is <30.43 weeks. [29]ur research identified that unfortunately, not all patients benefited from the treatment, but only 62% of them, and the preferred scheme was R-CHOP (44%).In our study, the R-CHOP treatment scheme was especially associated with the low age of the patients (P = .034),but also with the nodal localization of the lymphoma (P = .004).Both R-CHOP and CHOP protocol schemes were associated with a good performance status ECOG (P < .001),with a low Ann Arbor stage (P = .005),and with a favorable IPI risk score (P < .001).Also, in the current study, the most effective treatment for survival was the use of the CHOP scheme, in its case an average survival of 2323.32 days (331.90 weeks) was observed, while in the R-CHOP scheme, an average of 2278.90 days (325.55 weeks) was observed, and those who did not perform chemotherapy treatment had an average of 117.16 days (16.73 weeks), (P < .001).The lack of chemotherapeutic treatment represents a risk factor regarding patient mortality (HR = 6.750,P < .001)(see Table 2).
Research studies by Huang et al and Scherer et al have found that LDH is a useful indicator of the size of the tumor in solid and hematological malignancies. [30,31]The LDH value serves as a valuable indicator of the severity of the illness and the efficacy of therapy and is one of the components of the International Prognostic Index (IPI).However, a few studies have shown that in NHL lymphomas, LDH correlates strongly with higher levels of cell-free tumor DNA and might be a surrogate of increased circulating tumor cells. [31,32]The present study demonstrates that in 56% of cases, a value above the normal limit (>225) was observed and it had an average value of 471.04 UI/L.Regarding the association between the laboratory tests, we observed an increased LDH at admission that was statistically significantly associated with the presence of anemia (P = .033),and also with low hemoglobin values (P = .011).Also, we observed a statistically significant association between the LDH values and hemoglobin-the higher the LDH value, the lower the hemoglobin (P = .001).However, in the case of increased LDH values, the ECOG performance status was low (0-1), (P = .033).
In our research, regarding the survival of the patients according to this parameter, an increased LDH value was associated with low survival of the patients, 869.34 days (124.19 weeks) versus 2447.10 days (349.58 weeks), (P = .002),(Fig. 2).An elevated LDH value at admission represented an independent negative risk factor in terms of patient survival (HR = 3.100, P = .004)(Fig. 5 and Table 2).
In a research study by Prochazka et al, higher uric acid levels were linked to lower PFS (hazard ratio [HR]) and overall survival (OS) in univariable time-to-event analysis. [33]yperuricemia (20%) was one of the most prevalent comorbidities in our study group, although it had no relationship to a worse OS.
In human ABC DLBCL cell lines, MYD88 forms a complex with the IL-1 receptor-associated kinase 1 and IL-1 receptor-associated kinase 4 and promotes NF-κB and Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signaling, resulting in lymphoma cell survival. [9]fter the first report of highly oncogenic MYD88 L265P mutation in ABC DLBCL biopsies, [9] the L265P mutation was also detected in 5 cases (36%) of 14 primary central nervous system lymphoma (PCNSL) and 11 (69%) of 16 primary cutaneous DLBCL leg-type, respectively. [9,34,35]In our study, genetic analysis revealed a mutant status of MYD88 in 8% of patients and L265P mutation was observed in 4 of 40 (10%) DLBCL cases (Table 4).Also, none of the patients with the L265P mutation showed a significant association with clinical parameters of DLBCL, including the patient's age, sex, tumor location, ECOG performance status, LDH level, IPI score, and Ann Arbor stage.
Patients with GCB DLBCL demonstrated a lowered mutation frequency, whereas patients with non-GCB DLBCL exhibited a mutant status of MYD88 in 27.8% of cases.These results are consistent with other studies' findings that the prevalence of MYD88 L265P mutations in DLBCL patients ranged from 6.5% to 19%. [5,9,36,37]In our study, the mutation frequency was similar between GCB and non-GCB DLBCL groups.
Overall, the subtypes of DLBCL still represent a heterogeneous group of neoplasms when the presence of MYD88 L265P mutation is taken into account. [1]anger sequencing further confirmed the presence of MYD88 L265P in patients with mature B-cell NHL.Depending on the laboratory circumstances, both analytical approaches might be employed in clinical testing situations because the results were the same as those obtained by sequencing analysis.However, it should be highlighted that in FFPE samples with fragmented nucleic acids, Sanger sequencing may not be able to detect lower-frequency mutations and may result in inadequate PCR amplification templates. [1]f the 4 MYD88 L265P mutations, 2 had nodal involvement, while the other 2 had extranodal involvement, including immune-privileged sites (brain).The patients with extranodal involvement were in the advanced stage (Ann Arbor stage III-IV).Since the MYD88 mutation is present in several B-cell lymphomas, it has been hypothesized that the MYD88 mutation represents an early molecular event of lymphomagenesis. [38]A study by Fujiishi et al did not detect the mutation in a diagnostic biopsy specimen of a patient with DLBCL, although the postmortem sample of a patient with DLBCL tested positive for the mutation.They hypothesized that the mutation could be linked to a more aggressive phenotype. [39]According to our findings, individuals with the MYD88 L265P mutation were characterized by being at an advanced stage, which raises the possibility that the mutation may be crucial in the development of lymphoma and be linked to a worse prognosis for the disease.
According to a study by Kraan et al, MYD88 mutations were relatively uncommon in activated B-cell-like (ABC) DLBCLs www.md-journal.comarising from the lymph nodes or intestine; however, the mutation rate was higher in tumors originating from immuneprivileged sites. [40]Also, other studies have indicated that MYD88 mutations were more prevalent in lymphomas of the primary central nervous system. [34,41]n contrast, in our research the MYD88 L265P mutation was detected in half of the patients with non-GCB DLBCL with nodal involvement, while the remaining mutation was present in an immune-privileged site: the CNS (brain).Similarly, only half of the patients with GCB DLBCL with nodal involvement showed MYD88 L265P mutation.
As far as we are aware, only Choi et al and Caner et al evaluated MYD88 expression by IHC analysis in DLBCL.The study of Choi et al did not identify any associations between MYD88 expression and clinicopathological variables such as stage or IPI score. [5]In contrast, the study of Caner et al found a reverse relationship between MYD88 overexpression and stage and IPI score. [1]In our research, we also analyzed MYD88 protein expression by IHC analysis in mature B-cell NHLs, and similar to the results reported by Choi et al and by Caner et al, our study failed to reveal the correlation between MYD88 mutation and expression.A potential limitation of our study is that we focused solely on MYD88 L265P, the most prevalent mutation found in lymphomas; other MYD88 variants, such as S222R and T294P, could be responsible for the altered protein expression.Therefore, more research is required to understand other genetic and epigenetic changes that underlie the MYD88 expression found in several B-cell NHL patients.
According to studies by Coiffier et al and Fu et al, R-CHOPtreated patients with DLBCL are known to have better survival than CHOP-treated patients. [6,42]n other studies by Ruiz-Delgado et al and Winter et al, the group treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and the CHOPtreated group showed no significant difference in disease-free survival and OS. [43,44]In contrast, in our cohort, the group of patients treated with CHOP had better survival than R-CHOPtreated patients, 2323.32 days (331.90 weeks) versus 2278.90 days (325.55 weeks), but similar to other studies, showed no significant difference in OS.
According to a study by Pasqualucci et al, they have reported that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/ TTF (ARHH), and PAX5, in more than 50% of diffuse largecell lymphomas, which are tumors derived from germinal centers. [45]Overall, the results indicate that the hypermutation of PIM1, MYC, RhoH/TTF, and PAX5 is not common to all germinal-center-derived tumors, but is instead largely restricted to DLBCL and alter-natively, different mutated genes may be selected in different lymphoma types. [45]n another study by Zhou et al, the most frequently mutated genes in their patient cohort diagnosed with non-Hodgkin lymphoma were PIM1 (77.27%),MYD88 (63.64%),CD79B (59.09%), and KMT2D (50.0%). [15]In our study, PIM1 p.G28A mutation was observed in 2% of NHL cases and 1 of 4 (25%) THRLBCL cases.
According to Zhou et al, PIM1 and MYD88 were highly expressed in the patient's cohort and were related to their OS time, and the high expression of PIM1 or MYD88 was correlated with a higher risk score, and the high expression of MYD88 was also correlated with the elevated level of LDH.However, there was no significant correlation with patient age, gender, and/or type. [15]Also, the same research report has shown that multivariate Cox regression model analysis including risk score, LDH level, treatment method, and PIM1 and MYD88 expression status indicated that the expression status of MYD88 was an independent predictor of OS with a HR of 0.004. [15]Consistent with those studies, in our case multivariate analysis observed that the association between a high LDH at admission and the immunohistochemical expression of PIM1 or with the mutant status of the PIM1 gene represents negative prognostic factors (HR = 2.066, P = .042,respectively HR = 3.100, P = .004)(see Table 2).

Conclusions
In conclusion, our study shows that, compared to previously reported studies, the incidence of MYD88 L265P and PIM1 p.G28A mutations in patients with DLBCL is lower and give a clear picture of the mutational landscape in DLBCL, which may lead to novel ideas for treating the condition.
Thus, regardless of the MYD88 L265P mutation, MYD88 expression may have significant implications on the progression of DLBCL, independent of MYD88 L265P mutation.
Because of this, we suggest that MYD88 expression and the L265P mutation should only be utilized as prognostic indicators for instances of advanced-stage diseases.Independent of the mutation, MYD88 expression does not indicate the prognosis of the illness.
Regarding PIM1, because of the association between a high LDH at admission and the immunohistochemical expression of PIM1 or with the mutant status of the PIM1 gene, we hypothesized that both immunohistochemical and expression of PIM1 could be used as prognostic factors in patients diagnosed with large B-cell lymphomas.

Figure 2 .
Figure 2. Univariate Cox regression analysis that expresses an elevated LDH value at admission represented an independent negative risk factor in terms of patient survival (HR = 3.100, P = .004).

Figure 3 .
Figure 3. Kaplan-Meier survival graphic that shows a lower survival in the testis and spleen localizations of lymphomas (P = .010).

Figure 4 .
Figure 4. Kaplan-Meier survival graphic that shows a lower survival in the extranodal localizations of lymphomas (P = .130).

Figure 5 .
Figure 5. Univariate Cox regression analysis showed that the ECOG performance status represented an independent risk factor predicting mortality (HR = 9.372, P < .001).

Figure 6 .
Figure 6.Univariate Cox regression analysis showed that the high risk represented by the IPI score represents a negative risk factor in terms of patient survival (HR = 4.654, P < .001).

Figure 7 .
Figure 7. (A-D) Immunohistochemistry evaluation of surgical specimens: (A) the image shows a positive control immunostain using a sample of kidney tissue for MYD88 biomarker (IHC; 40×); (B) the image shows a positive control immunostain using a sample of tonsil tissue for PIM1 biomarker (IHC; 40×); (C) cytoplasmic expression of MYD88 biomarker was positive in scattered cells within the tumor (IHC; 40×); (D) nuclear expression of PIM1 biomarker was positive in most of the cells within the proliferation (IHC; 40×).

Table 2
Univariate and multivariate Cox regression analyses.

Table 3
The distribution of demographic aspects, tumor characteristics, and personal history are stratified according to diagnosis.